WO2016107112A1 - 一种富含胶原蛋白的人工神经支架及其制备方法 - Google Patents

一种富含胶原蛋白的人工神经支架及其制备方法 Download PDF

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WO2016107112A1
WO2016107112A1 PCT/CN2015/082178 CN2015082178W WO2016107112A1 WO 2016107112 A1 WO2016107112 A1 WO 2016107112A1 CN 2015082178 W CN2015082178 W CN 2015082178W WO 2016107112 A1 WO2016107112 A1 WO 2016107112A1
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collagen
ngf
stent
artificial nerve
scaffold
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French (fr)
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李扬德
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东莞颠覆产品设计有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body

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  • the present invention relates to the field of nerve repair technology, and in particular to a collagen-rich artificial nerve stent and a preparation method thereof.
  • Peripheral nerve damage or breakage due to various trauma causes a decrease or loss of sensory and motor function. Severe peripheral nerve injury often leads to paralysis or permanent loss of labor. Peripheral nerve injury has a high incidence in China. According to statistics, in trauma patients, nerve injury of the extremities accounts for about 10% of the total number of trauma, and about 60% of the fractures of firearm injuries have neurological injuries. To this end, we need to find the ideal treatment for peripheral nerve injury. For peripheral nerve fractures, if the fracture gap is large, the fractured peripheral nerve must be regenerated and repaired by bridging the graft.
  • the present invention provides an artificial nerve scaffold rich in collagen and a preparation method thereof.
  • the artificial nerve scaffold prepared by the method has good mechanical properties, is biodegradable, and has a three-dimensional structure required for nerve regeneration.
  • NGF has far-reaching significance for nerve repair.
  • a collagen-rich artificial nerve stent comprising a stent film and a degradable metal wire wrapped by a stent film, the main component of which is collagen, nerve growth factor (NGF) And polylactic acid-polyglycolic acid copolymer (PLGA).
  • NGF nerve growth factor
  • PLGA polylactic acid-polyglycolic acid copolymer
  • the mass ratio of the polylactic acid-polyglycolic acid copolymer to the collagen is 8-7:2-3.
  • the NGF and gelatin are made into NGF gelatin microspheres distributed in the scaffold film, and the mass content of the NGF gelatin microspheres is 20-40% of the total mass content of the PLGA powder and the collagen powder.
  • the main component of the degradable metal wire is magnesium, zinc, calcium, iron or an alloy thereof.
  • the stent is in the form of a sheet, a strip or a cylinder.
  • the degradable wires are distributed transversely and longitudinally along the stent.
  • the preparation method of the collagen-rich artificial nerve stent comprises the following steps:
  • NGF gelatin microspheres were prepared by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro. ball;
  • stent film The PLGA powder and the collagen powder obtained in the step 1) are weighed according to the mass ratio of 8-7:2-3, the PLGA powder is completely dissolved in the dichloromethane, the collagen powder is added, and the step 2) is obtained. NGF gelatin microspheres, the ultrasonic dispersion is uniform, and the stent film material is obtained;
  • step 4) transferring the scaffold film obtained in step 3) into the mold cavity of the film forming mold, and placing the straightened degradable metal wire in the mold cavity in the transverse direction and the longitudinal direction in the molding direction of the stent film, so that the degradable metal wire is immersed in the stent film
  • the solvent is naturally evaporated and dried, and vacuum is applied for 48 hours. At this time, the dried stent film is wrapped around the degradable metal wire to obtain the artificial nerve stent.
  • the particle size of the NGF gelatin microspheres was 1-20 ⁇ m, and the sealing ratio of the microspheres to NGF was 81.5%.
  • the NGF gelatin microspheres are used in an amount of 20-40% based on the total amount of the PLGA powder and the collagen powder.
  • the main component is collagen, which also contains fibronectin, laminin, aminodextran, proteoglycan, etc., which are beneficial to nerve conduction. And the growth of axons, with good biocompatibility.
  • the use of ECM alone for nerve injury transplantation will make it difficult to provide morphological support for nerve growth because ECM is too soft, and at the same time, these ECMs will be calcified in a short time. Therefore, the combination of collagen and PLGA allows the resulting scaffold to have certain mechanical properties, provide morphological support for nerve growth, and prevent collagen calcification.
  • scanning electron fiber mirrors show that the internal structure of the obtained artificial nerve scaffold is honeycomb, which provides space for the nerve cells to crawl and grow.
  • Degradable metals and their alloys can guide the growth of nerve cells, which is beneficial to the repair of nerve cells.
  • NGF is a biologically active substance. During the process of peripheral nerve repair, nerve tissue secretes NGF. Adding exogenous NGF is beneficial to nerve repair.
  • NGF gelatin microspheres are prepared by using gelatin. Since gelatin is a water-soluble substance, it is not dissolved in an organic solvent. In the preparation of the material, gelatin can isolate NGF from the organic solvent, thereby facilitating the maintenance of NGF activity. Gelatin also contributes to the slow release of the NGF.
  • the acidic substances produced by the degradation of PLGA cause local acid accumulation and affect the stability of the combination. Therefore, this can be improved by adding a weakly basic additive.
  • the weakly basic additive is further added in step 3).
  • the weakly alkaline additive is a basic amino acid, chitosan, sodium alginate or sodium hydrogencarbonate, and the amount thereof is such that the pH of the degradation environment of the PLGA is 7.28-6.62.
  • the beneficial effects of the invention are: mechanical properties of the collagen-rich artificial nerve scaffold of the invention Good, biodegradable, with the three-dimensional structure and NGF required for nerve regeneration, has far-reaching significance for nerve repair.
  • FIG. 1 is a schematic view showing the structure of a collagen-rich artificial nerve stent of the present invention.
  • Fig. 2 is a three-dimensional structure of a porous structure formed by decellularization of pig skin in the present invention.
  • Figure 3 is a view of the morphology of the NGF gelatin microspheres obtained in the present invention under a microscope.
  • Figure 4 is a cross section of the artificial nerve stent obtained by scanning electron microscopy.
  • a collagen-rich artificial nerve stent comprises a stent film 1 and a degradable metal wire 2 wrapped by a stent film 1.
  • the stent can be prepared into various shapes according to the needs of use, and is generally in the form of a sheet, a strip or a cylinder.
  • Figure 1 shows a cylindrical shape.
  • the degradable wire 2 is distributed transversely and longitudinally along the stent.
  • the scaffold film 1 contains collagen, nerve growth factor and polylactic acid-polyglycolic acid copolymer (PLGA); the mass ratio of the polylactic acid-polyglycolic acid copolymer to the collagen is 8-7:2
  • the mass content of the NGF gelatin microspheres is 20-40% of the total mass content of the PLGA powder and the collagen powder.
  • the main component of the degradable metal wire 2 is magnesium, zinc, calcium, iron or an alloy thereof.
  • a method for preparing an artificial nerve scaffold rich in collagen comprising the following steps:
  • the resulting collagen powder was observed by H.E staining and scanning electron microscopy, as shown in FIG. It can be seen that the pig skin is decellularized to form a loose porous three-dimensional structure.
  • NGF gelatin microspheres were prepared by NGF was added to a 20% aqueous gelatin solution, the concentration of NGF was 0.06-0.10 ⁇ g/ml, and the mixed solution was added to a salad oil of 3.5 times its volume, and preheated to 37 ° C.
  • the oil-water emulsion was emulsified by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro. ball.
  • the morphology of the obtained NGF gelatin microspheres was observed with a microscope as shown in FIG.
  • the NGF gelatin microspheres have a particle diameter of 1-20 ⁇ m.
  • stent film The PLGA powder and the collagen powder obtained in the step 1) are weighed according to the mass ratio of 8-7:2-3, the PLGA powder is completely dissolved in the dichloromethane, the collagen powder is added, and the step 2) is obtained. NGF gelatin microspheres, the ultrasonic dispersion is uniform, and the stent film material is obtained;
  • step 4) Transfer the scaffold film obtained in step 3) into the mold cavity of the film forming mold, and place the straightened and degradable metal wire in the mold cavity along the direction of the stent film forming direction, so that the degradable metal wire is immersed in the scaffold film.
  • the solvent is naturally volatilized and dried, and vacuumed for 48 hours. At this time, the dried stent film wraps the degradable metal wire to obtain the artificial nerve stent.
  • a method for preparing an artificial nerve scaffold rich in collagen comprising the following steps:
  • NGF gelatin microspheres were prepared by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro. ball.
  • Step 3 Preparation of stent film: The PLGA powder and the collagen powder obtained in the step 1) are weighed according to the mass ratio of 8-7:2-3, the PLGA powder is completely dissolved in the dichloromethane, and the collagen powder and the weak alkaline additive are added. Step 2) The obtained NGF gelatin microspheres are uniformly dispersed by ultrasonic to obtain a scaffold film; the weakly alkaline additive is a basic amino acid, and the added amount thereof is such that the pH of the degradation environment of the PLGA is 7.28-6.62;
  • step 4) Transfer the scaffold film obtained in step 3) into the mold cavity of the film forming mold, and place the straightened and degradable metal wire in the mold cavity along the direction of the stent film forming direction, so that the degradable metal wire is immersed in the scaffold film.
  • the solvent is naturally volatilized and dried, and vacuumed for 48 hours. At this time, the dried stent film wraps the degradable metal wire to obtain the artificial nerve stent.
  • the cross section of the artificial nerve stent obtained by scanning electron microscopy showed that the artificial nerve stent obtained had a honeycomb structure in cross section.
  • the honeycomb structure provides the required voids for the creep and extension of nerve cells.

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Abstract

一种富含胶原蛋白的人工神经支架及其制备方法，所述神经支架包括支架膜料(1)以及支架膜料(1)包裹的可降解金属丝(2)。所述支架膜料(1)含有胶原蛋白，神经生长因子(NGF)和聚乳酸-聚羟基乙酸共聚物(PLGA)。所述制备方法包括制备胶原蛋白粉末，制备NGF明胶微球，制备支架膜料(1)以及用支架膜料(1)包裹可降解金属丝(2)的步骤。

Description

一种富含胶原蛋白的人工神经支架及其制备方法 技术领域
本发明涉及神经修复技术领域，具体地，涉及一种富含胶原蛋白的人工神经支架及其制备方法。
背景技术
由于各种外伤原因导致的周围神经损伤或断裂造成伤者感觉和运动功能的下降或丧失。严重的外周神经损伤往往导致患者瘫痪或永久丧失劳动力。外周神经损伤在我国发病率较高，据统计，在外伤患者中，四肢神经伤约占外伤总数的10％，火器伤骨折中约有60％的合并神经伤。为此，需寻找理想的外周神经伤治疗手段。对于外周神经断裂类损伤，若断裂缺口较大，就须借助对缺口进行桥接移植手术才能使断裂的周围神经得到再生修复。
目前，常用的移植体有自体移植体和人工神经支架。自体移植体来源有限且会带来供体后遗症。人工神经支架方面，由于非降解导管材料可能导致神经压迫和二次手术的风险，近年来研究的主要趋向于生物可降解支架材料的开发和研究。当前，美国已经批准多种品牌，如，NeuraGen、SaluBridge，Neurolac等的神经导管用于临床。然而，我国在神经支架方面的研究较为落后，并没有开发出具有自主知识产权的神经导管。
发明内容
为了克服现有技术的不足，本发明提供了一种富含胶原蛋白的人工神经支架及其制备方法，该方法制备的人工神经支架力学性能良好，生物可降解，具有神经再生所需要的三维结构和NGF，对神经修复具有深远的意义。
本发明的技术方案如下：一种富含胶原蛋白的人工神经支架，包括支架膜料以及支架膜料包裹的可降解金属丝，所述支架膜料的主要成分为胶原蛋白，神经生长因子(NGF)和聚乳酸-聚羟基乙酸共聚物(PLGA)。
所述聚乳酸-聚羟基乙酸共聚物和所述胶原蛋白的质量比为8-7∶2-3。
所述NGF与明胶制成NGF明胶微球分布与所述支架膜料中，所述NGF明胶微球的质量含量为PLGA粉末和胶原蛋白粉末总质量含量的20-40％。
所述可降解金属丝的主要成分为镁，锌，钙，铁或其合金。
所述支架为片状，长条状或圆柱状。
所述可降解金属丝沿所述支架横向和纵向分布。
所述富含胶原蛋白的人工神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂18-36h；用清水漂洗三次，每次18-24h；于0.5％的胰蛋白酶中在37℃振荡浸泡18-30h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48-72h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白；
2)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；
3)制备支架膜料：按质量比例8-7∶2-3称量PLGA粉末和步骤1)所得胶原蛋白粉末，将PLGA粉末完全溶解在二氯甲烷中，加入胶原蛋白粉末和步骤2)所得NGF明胶微球，超声分散均匀，得到支架膜料；
4)将步骤3)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜 料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述人工神经支架。
所述NGF明胶微球的粒经为1-20μm，微球对NGF的封载率为81.5％。
所述NGF明胶微球的用量为PLGA粉末和胶原蛋白粉末总量的20-40％。
动物皮被处理后，脱出了细胞，形成了结构松散的ECM，其主要成分是胶原蛋白，还含有纤维连接蛋白，层粘连接蛋白，氨基葡聚糖，蛋白聚糖等，这些有利于神经传导以及轴突的生长，具有良好的生物相容性。将ECM单独用于神经损伤移植，会由于ECM太软，难以为神经生长提供形态学上的支撑，同时，短时间内，这些ECM就会被钙化。因此，将胶原蛋白与PLGA结合使用使得制成的支架具有一定的力学性能，能够为神经生长提供形态学上的支撑，也防止胶原蛋白钙化。另外，扫描电子纤维镜表明，所得人工神经支架的内部结构为蜂窝状，为神经细胞的爬行和生长提供了空间。
可降解金属及其合金可以引导神经细胞生长，从而有利于神经细胞的修复。
NGF是生物活性物质，在周围神经修复的过程中，神经组织会分泌NGF，添加外源的NGF有利于神经的修复。本发明中，使用明胶制备NGF明胶微球，由于明胶是水溶性物质，在有机溶剂中不溶解，在材料的制备中明胶可将NGF与有机溶剂隔离，从而有利于保持NGF活性。明胶还有助于所述NGF的缓慢释放。
PLGA的降解所产生的酸性物质会造成局部集酸现象，影响组合的稳定性。因此，可以通过添加弱碱性添加剂改善这一情况。较佳地，步骤3)中还添加有所述弱碱性添加剂。所述弱碱性添加剂为碱性氨基酸，壳聚糖，海藻酸钠或碳酸氢钠，其添加量应保证所述PLGA的降解环境pH值为7.28-6.62。
本发明的有益效果为：本发明所述富含胶原蛋白的人工神经支架力学性能 良好，生物可降解，具有神经再生所需要的三维结构和NGF，对神经修复具有深远的意义。
附图说明：
图1为本发明所述富含胶原蛋白的人工神经支架的结构示意图。
图2为本发明中猪皮经脱细胞处理后形成的疏松多孔的三维结构。
图3为显微镜下本发明中所得NGF明胶微球的形态
图4为用扫描电镜观测所得人工神经支架的横断面。
具体实施方式
下面结合附图和优选实施例对本发明作进一步的描述，但本发明的实施方式不限于此。
参照图1，一种富含胶原蛋白的人工神经支架，包括支架膜料1以及支架膜料1包裹的可降解金属丝2。所述支架可以根据使用需要制备成各种形状，一般为片状，长条状或圆柱状。图1所示为圆柱状。所述可降解金属丝2沿所述支架横向和纵向分布。
所述支架膜料1含有胶原蛋白，神经生长因子和聚乳酸-聚羟基乙酸共聚物(PLGA)；所述聚乳酸-聚羟基乙酸共聚物和所述胶原蛋白的质量比为8-7∶2-3；所述NGF明胶微球的质量含量为PLGA粉末和胶原蛋白粉末总质量含量的20-40％。
所述可降解金属丝2的主要成分为镁，锌，钙，铁或其合金。
实施例1：
一种富含胶原蛋白的人工神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：猪皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂24h；用清水漂洗三次，每次20h；于0.5％的胰蛋白酶中在37℃振荡浸泡24h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡50h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白。
用H.E染色及扫描电镜观测所得胶原蛋白粉末，如图2所示。经观察可知，猪皮经脱细胞处理后形成疏松多孔的三维结构。
2)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球。用显微镜观察所得NGF明胶微球的形态，如图3所示。所述NGF明胶微球的粒经为1-20μm。
3)制备支架膜料：按质量比例8-7∶2-3称量PLGA粉末和步骤1)所得胶原蛋白粉末，将PLGA粉末完全溶解在二氯甲烷中，加入胶原蛋白粉末，步骤2)所得NGF明胶微球，超声分散均匀，得到支架膜料；
4)将步骤3)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述人工神经支架。
实施例2：
一种富含胶原蛋白的人工神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：猪皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂36h；用清水漂洗三次，每次24h；于0.5％的胰蛋白酶中在37℃振荡浸泡18h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白。
2)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球。
3)制备支架膜料：按质量比例8-7∶2-3称量PLGA粉末和步骤1)所得胶原蛋白粉末，将PLGA粉末完全溶解在二氯甲烷中，加入胶原蛋白粉末和弱碱性添加剂，步骤2)所得NGF明胶微球，超声分散均匀，得到支架膜料；所述弱碱性添加剂为碱性氨基酸，其添加量应保证所述PLGA的降解环境pH值为7.28-6.62；
4)将步骤3)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述人工神经支架。
用扫描电镜观测所得人工神经支架的横断面显示所得人工神经支架的横断面呈蜂窝状结构。蜂窝状结构为神经细胞其中的爬行及延伸提供了需要的空隙。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不 能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其架构形式能够灵活多变，可以派生系列产品。只是做出若干简单推演或替换，都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。

Claims (10)

  1. 一种富含胶原蛋白的人工神经支架，其特征在于，包括支架膜料以及支架膜料包裹的可降解金属丝，所述支架膜料的主要成分为胶原蛋白，神经生长因子(NGF)和聚乳酸-聚羟基乙酸共聚物(PLGA)。
  2. 如权利要求1所述的富含胶原蛋白的人工神经支架，其特征在于，所述聚乳酸-聚羟基乙酸共聚物和所述胶原蛋白的质量比为8-7∶2-3。
  3. 如权利要求1所述的富含胶原蛋白的人工神经支架，其特征在于，所述NGF与明胶制成NGF明胶微球分布与所述支架膜料中，所述NGF明胶微球的质量含量为PLGA粉末和胶原蛋白粉末总质量含量的20-40％。
  4. 如权利要求1所述的富含胶原蛋白的人工神经支架，其特征在于，所述可降解金属丝的主要成分为镁，锌，钙，铁或其合金。
  5. 如权利要求1所述的富含胶原蛋白的人工神经支架，其特征在于，所述支架为片状，长条状或圆柱状。
  6. 如权利要求1所述的富含胶原蛋白的人工神经支架，其特征在于，所述可降解金属丝沿所述支架横向和纵向分布。
  7. 一种富含胶原蛋白的人工神经支架的制备方法，其特征在于，包括如下步骤：
    1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂18-36h；用清水漂洗三次，每次18-24h；于0.5％的胰蛋白酶中在37℃振荡浸泡18-30h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48-72h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；
    2)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；
    3)制备支架膜料：按质量比例8-7∶2-3称量PLGA粉末和步骤1)所得胶原蛋白粉末，将PLGA粉末完全溶解在二氯甲烷中，加入胶原蛋白粉末和步骤2)所得NGF明胶微球，超声分散均匀，得到支架膜料；
    4)将步骤3)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述人工神经支架。
  8. 如权利要求7所述的富含胶原蛋白的人工神经支架的制备方法，其特征在于，所述NGF明胶微球的粒经为1-20μm，微球对NGF的封载率为81.5％。
  9. 如权利要求7所述的富含胶原蛋白的人工神经支架的制备方法，其特征在于，所述NGF明胶微球的用量为PLGA粉末和胶原蛋白粉末总量的20-40％。
  10. 如权利要求7所述的富含胶原蛋白的人工神经支架的制备方法，其特征在于，步骤3)中还添加有所述弱碱性添加剂。所述弱碱性添加剂为碱性氨基酸，壳聚糖，海藻酸钠或碳酸氢钠，其添加量应保证所述PLGA的降解环境pH值为7.28-6.62。
PCT/CN2015/082178 2014-12-29 2015-06-24 一种富含胶原蛋白的人工神经支架及其制备方法 WO2016107112A1 (zh)

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